Ultra-high yield intravenous immune globulin preparation

ABSTRACT

An efficacious large-scale alcohol-free plasma fractionation production process which produces a high-yielding, non-denatured, double viral-inactivated intravenous human immune gamma globulin (IgG) product. The process employs sodium citrate in two initial fractionation steps, followed by diafiltration to remove sodium citrate.

CONTINUATION-IN-PART

This application for patent is a Continuation-in-Part of U.S. patentapplication Ser. No. 11/217,956, titled AN ULTRA-HIGH YIELD INTRAVENOUSIMMUNE GLOBULIN PREPARATION and filed Sep. 1, 2005.

FIELD OF INVENTION

This invention relates generally to methods for immune serum globulinpurification, and, more particularly, to methods for alcohol-freeseparation of immune globulin from blood plasma or other blood basedmaterial.

BACKGROUND AND DESCRIPTION OF RELATED ART

Commonly, contemporary methods for separation of immune globulins (IgG)from blood plasma or other blood based material depend upon early workby Edwin J. Cohn. As found in U.S. Pat. No. 5,177,194 issued Jan. 5,1993 to Maria E. Sarno, et al. (SARNO), “One scheme in widespread use isthe well-known Cohn fractionation method, which is based on differentialprecipitation using cold ethanol.” Cohn et al. J. Am. Chem. Soc. 68, 459(1946).

A U.S. Pat. No. 2,390,074 issued Dec. 4, 1945 to Edwin J. Cohn (Cohn)disclosed use of alcohol, acetone and dioxane as precipitants in suchfractionation processes. Continued dependence upon alcohol as aprecipitant is further demonstrated in U.S. Pat. No. 6,893,639 B2 issuedMay 17, 2005 to Joshua Levy, et al. (Levy), wherein it is stated, “Theconventional industrial methods of immune globulins purification fromblood plasma are based on cold ethanol fractionation whichco-precipitate groups of proteins based on their isoelectric points atgiven alcohol concentration at sub-zero temperatures.”

Cohn's work was stimulated by the need of the military for a stablesolution for use as a plasma volume expander during World War II toreplace lyophilized plasma. Consequently, the Cohn method focused onoptimizing the process for separating the albumin fraction whichprovides the osmolality necessary for plasma volume expansion.

Even so, the use of alcohol precipitants is not without difficulties, asillustrated by Cohn, “Some protein precipitants, such as alcohol, have atendency to denature many proteins with which they come in contact, thedanger of denaturation increasing with concentration of the alcohol andincrease in temperature. For many proteins, it has been found advisableto exercise considerable care in mixing the precipitant with the plasmaor other protein solution in order to avoid denaturation of theprotein.” For this reason, it is considered prudent to provide analcohol free method for blood plasma and other blood based materialfractionation, including IgG purification.

In the 1970's, chromatography was found to be useful in the separationand purification of plasma proteins. Chromatography separates plasmaproteins by specifically targeting unique characteristics of each,including molecular size (gel filtration), charge (ion exchangechromatography), and known interactions with specific molecules(affinity chromatography).

The use of various chromatographic methods on an industrial scale hasbeen adopted for the isolation of small-weight, high-value proteins,such as Factor VIII, from plasma, and for the final purification ofgamma globulin after separation from the plasma by Cohn, or modifiedCohn methodologies. However, the separation of the large-weight,lower-value fractions such as albumin and gamma globulin, on anindustrial scale has not been found to be practical.

Two U.S. Patent Applications, filed by Edward Shanbrom, havingApplication Numbers 20030022149 (Shanbrom '149) and 20030129167(Shanbrom '167) filed Jan. 30, 2003 and Jul. 10, 2003, respectively,teach of use of carboxylic salts (e.g., trisodium citrate) as an agentfor enhancing formation of a cryoprecipitate from plasma. The method(s)of Shanbrom generally involve trisodium citrate and other citrate saltsas agents for enhancing production of blood clotting factors fromcryoprecipitate.

Shanbrom '149 teaches in paragraph 0009 that “It is an object of thepresent invention to provide enhanced yields of cryoprecipitate.”Shanbrom also teaches, in paragraph 0011, that carboxylic acids areeffective agents for enhancing the production of blood clotting factorsfrom the cryoprecipitate. Shanbrom '149 notes that the addition ofcitrate to plasma, especially at concentrations between two and tenpercent, by weight, does not appreciably denature labile proteins.Moreover, it is noted in Shanbrom '149 that citrate potentiates orenhances the killing of microorganisms by heat treatment.

Shanbrom '167 notes in paragraph 0015 that “Not only does added citrateincrease the amount of cryoprecipitate, it simplifies the process bydecreasing the requirement for freezing” plasma in order to harvestcryoprecipitate. Shanbrom clearly teaches use of production of acryoprecipitate for the purpose of fractionating products from thecryoprecipitate through the use of trisodium citrate in concentrationsof two to ten percent.

While Shanbrom '149 and '167 deal directly with extracting labilecoagulation products from a cryoprecipitate formed through use ofcitrate compounds, particularly trisodium citrate, and with killingmicroorganisms in the cryoprecipitate using the citrate compounds, theinstant invention deals directly with extracting non-labile products(e.g., albumin, gamma globulin and alpha-1-antitrypsin) from asupernatant formed through use of citrate compounds. Shanbrom neitherteaches nor addresses using a supernatant in any way.

In the 1950's, it was discovered that a “cryoprecipitate” derived fromblood based material, contained various factors was useful in treatingclotting disorders such as hemophilia. Such a cryoprecipitate, as thename implies, was obtained by freezing blood plasma followed bycontrolled thawing at zero to four degrees centigrade to form a liquidsuspension of the precipitate. Such a cryoprecipitate was then availablefor fractionation using methods according to Cohn to produce albumin andgamma globulin. Subsequent developments led to fractionation ofcryoprecipitate into pure concentrates of Factor VIII, von WillebrandFactor, and other clotting factors. Such may be accomplished by usingnon-alcohol separations and chromatographic purification.

BRIEF SUMMARY AND OBJECTS OF THE INVENTION

In brief summary, this instant invention provides novel and effectivemethods of isolating gamma globulin from plasma and formulating it intoan intravenous injectable preparation. Accordingly, this invention,which may be defined to be “an ultra-high yield intravenous immuneglobulin preparation,” achieves higher yields of a superior qualitygamma globulin by directly and expeditiously separating the gammaglobulin from the plasma by means of a non-denaturing precipitant,sodium citrate.

The inventive process is for fractionating blood-based products toproduce a useful, non-denatured immunoglobulin (IgG) product whichinvolves the following critical steps:

(a) adding a first volume of sodium citrate to a quantity of blood-basedmaterial to be fractionated to form a supernatant product, free ofeuglobulins, the supernatant being separable from a residual paste andseparating the two;

(b) performing a second fractionation step by adding a second volume ofsodium citrate to the separated supernatant product, thereby forming asecond separable product which is a paste and a second residual productwhich is supernatant, such that the products may thereafter beseparated;

(c) forming a liquid dilution of the second separated paste product; and

(d) diafiltering the second separated and liquified paste product toform a low volume resulting product which is substantially free ofsodium citrate and ready for processing by currently known and practicedprocedures to complete production of the useful, non-denatured IgG.

In step (a), the first volume utilizes a fifty percent sodium citratesolution. The supernatant and residual paste should have a range ofeleven to thirteen percent, and preferably should approximate a twelvepercent concentration. If desired, the residual paste may be furtherfractionated into blood factors including VIII, IX, von Willebrand andfibrinogen. Separation of the products may be accomplished bycentrifuging or existing methods which are well known in chemistry art.

In step (b), the second volume utilizes addition of another fiftypercent sodium citrate solution. The second product is a paste and thesecond residual is a supernatant, both having a range of concentrationof approximately twenty-one to twenty-three percent sodium citrate,which should approximate twenty-two percent. As with step (a), ifdesired, the residual (supernatant) may be further processed into agroup of components comprising albumin and alpha-1-antitrypsin. Theproducts may be separated by centrifuging, filtering or other methodswhich are well-known in the chemistry art.

In step (c), it is preferred to dilute the paste product with waterhaving approximately four times the weight of the paste product,although other volumes of water may be judiciously selected within thescope of the invention.

In step (d) a diafiltration system with a 30 KD filtering membrane maybe used to separate the sodium citrate and excess water from theresulting product to permit further processing on an industrial scale.Note, that such filtering is made facile and possible by extractingeuglobulins from the supernatant in step (a). As used herein,euglobulins are defined to be those globulins which are insoluble inwater, but are soluble in saline solutions. Most importantly, ifeuglobulins are not removed from a solution and if the ionic strength ofthat solution is lowered towards deionized water (e.g., in the case ofthe instant invention), euglobulins foul a diafiltration system, therebyrendering it unuseable.

It is well-known that sodium citrate has long been used in lowconcentrations during the collection, preservation and storage of bloodplasma. Subsequent diafiltration after use of high concentrations ofsodium citrate as a precipitant substantially reduces the ionic strengthand volume of the gamma globulin solution, permitting the achievement ofchromatographic purification on an industrial scale.

Following separation of gamma globulin from plasma by this method,albumin and alpha-1-antitrypsin are subsequently removed from theremaining proteins by methods available from Cohn or others. Theprocess, according to the instant invention, enables the separation ofgamma globulin without exposing it to the denaturing effects of ethanolused in the Cohn process, hence leaving the gamma globulin in a nativestate. The denaturing effects of alcohol include the formation ofpolymers, aggregates and fragments of the gamma globulin molecule.However, the use of sodium citrate stabilizes the plasma while bringingabout precipitation of substantially all of the coagulation proteins,thus preventing the generation of enzyme activators and proteolyticenzymes.

The absence of the denaturing effects of ethanol, the stabilization ofthe plasma with sodium citrate, and the subsequent removal ofcoagulation proteins by means of sodium citrate results in a gammaglobulin preparation which has very low anti-complementary activity.

In summary, the process of the instant invention employs highconcentrations of sodium citrate combined with its subsequent removalfrom the gamma globulin concentrate by means of diafiltration, atechnique which became practical on an industrial scale in the 1980's.Final purification of the resulting gamma globulin is then practicallyand effectively achieved through the use of well-establishedchromatographic purification techniques. The invention reducesproduction costs as a result of higher yields, fewer fractionationsteps, shorter overall processing time, lower energy costs, and lowerchemical costs. Capital costs are less because of reduced spacerequirements, reduced work-in-process, reduced processing time, andelimination of the explosion proof environments required for ethanolprocessing.

Accordingly, it is a primary object to provide an effective intravenousgamma globulin preparation at a cost which is reduced from methods incurrent practice.

It is an important object to provide such a method and preparation whichis high-yielding.

It is a further object to provide a gamma globulin preparation which canbe rapidly infused with greater patient tolerance than gamma globulinproduced by methods employing alcohol.

It is therefore a principle object to provide an alcohol free method forpreparing gamma globulin.

It is an object to produce gamma globulin having reduced in-processformation of polymers, aggregates, fragments, enzyme activators andproteolytic enzymes compared with similar preparations produced usingtraditional alcohol based methods.

It is a further object to derive a cryoprecipitate as an optional methodaccording to the instant invention to form a liquid suspension of acryoprecipitate from which, through fractionation, Factor VIII, vonWillebrand Factor, and other clotting factors are produced.

These and other objects and features of the present invention will beapparent from the detailed description taken with reference toaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of a critical set of initial steps associatedwith the process according to the instant invention.

FIG. 1A is a flow diagram similar to the flow diagram of FIG. 1, buthaving additional steps which disclose an optional method for obtaininga cryoprecipitate as a paste which may be used to produce Factor VIII,von Willebrand Factor, and other clotting factors.

FIG. 2 is a flow diagram disclosing a series of steps which immediatelyfollow the steps seen in FIG. 1.

FIG. 3 is a flow diagram disclosing those procedural steps whichimmediately follow the steps seen in FIG. 2.

FIG. 4 is a flow diagram disclosing steps which immediately follow thesteps seen in FIG. 3 to provide a useful product.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Reference is now made to flow path elements illustrated in FIGS. 1-4.Generally, each rectangular box is used to illustrate a procedural step;each diamond is used to demonstrate a separation step; each ellipticalcylinder designates a product resulting from a preceding procedural orseparation step; and each circle is used to identify either a startingpoint or an off-sheet continuation path point.

Reference is now made to FIG. 1 wherein an initial portion 10-1 of anpreferred IgG process flow path, generally numbered 10, is seen. Asindicated after initial starting point 20, a volume of plasma 30 to beprocessed is selected for processing. It should be noted that whileplasma 30 is used by example in this description of an illustratedembodiment, other blood-based products may be processed within the scopeof the instant invention.

As part of procedure 40, selected frozen plasma 30 is warmed toapproximately five degrees Centigrade to form prepared plasma 50. Whilefive degrees is the target plasma 30 process temperature, which shouldbe maintained throughout the following steps in process 10, atemperature range between limits of two to eight degrees may be heldwithin the scope of the instant invention. Plasma 30 may be useddirectly if not selected in a frozen state (e.g., thawed during theprocess of removing a cryogenic precipitate by customary methods).

A batch of sodium citrate solution is made per procedure 52 wherein afifty percent sodium citrate solution is prepared by stirring fivehundred grams of sodium citrate into six hundred milliliters of purifiedwater. Stirring time should be thirty to sixty minutes or, alternately,until the sodium citrate is dissolved. At this point, dilute the mixturewith pure water to one thousand milliliters. Add a 50% citric acidsolution to the mixture until a pH of 7.0 is reached.

Preparatory to performing the first fractionation step (procedure 54), avolume of fractionation solution to be added to plasma 30 is calculated.It is a goal that the concentration of the sodium citrate fractionationsolution should be twelve percent. Also the pH of the fractionationsolution should be approximately 7.0. If necessary, adjust the pH to7.0.

The formula, (Formula I) for calculating respective volumes offractionation solution (sodium citrate) and plasma 30 are as follows:x=(C*V)/(0.5−C)

-   -   where:        -   x is desired volume of 50% sodium citrate solution;        -   C is a desired fractional concentration of sodium citrate;            (e.g., 0.12 or twelve percent): and        -   V is volume of solution to be diluted, (e.g., volume of            plasma 30).            An example of a calculation by Formula I is:

For a volume (V_(p)) of plasma 30 of 500 liters, and the desiredfractional concentration of sodium citrate is twelve percent:x=(0.12*500)/(0.5−0.12)=158 litersSolving Formula I for C yields Formula II into which values of volumesof plasma 30 and sodium citrate may be inserted as follows:C=(0.500*158)/(500+158)=0.12

For procedure 54, over a period of approximately five minutes, add theprepared sodium citrate fractionation solution (which may be at roomtemperature [i.e. approximately twenty degrees Centigrade]) to plasma 30(which has a starting temperature of five degrees Centigrade). Gentlystir while adding the sodium citrate solution. Once the sodium citratesolution is completely added to plasma 30, continue gently stirring theresulting slurry for approximately sixty minutes while reducing theslurry temperature to within a range of two to eight degrees centigrade.(The slurry should maintain pH at approximately 7.0 to 7.1.)

Upon completion of procedure 54, centrifuge as procedure 56. It isrecommended that a flow-through centrifuge (e.g., a WestphaliaCentrifuge) be used to separate component parts of the slurry into asupernatant liquid 60 and a paste 62 by normal procedures for thoseskilled in the art, while maintaining temperature of the slurry in therange of two to eight degrees Centigrade.

While supernatant liquid 60, which contains virtually all of the IgG ofthe original plasma, is retained for further processing as an integralpart of the instant inventive method, paste 62 may be further processedto recover blood factors, including Factors VIII, IX, von Willebrand andfibrinogen.

For the second fractionation phase, perform process step 64 which addsadditional sodium citrate fractionation solution to supernatant liquid60. Enough fifty percent sodium citrate is added to liquid 60 toincrease concentration of sodium citrate from twelve percent totwenty-two percent.

To calculate the volume of fifty percent sodium to be added, Formula IIIis provided as follows:C _(e)=((V ₆₀ *C ₆₀)+(V _(x) *C _(0.50)))/(V ₆₀ +V _(x))

-   -   Where C, is the desired end concentration of sodium citrate; V₆₀        is the volume of supernatant liquid 60; C₆₀ is sodium citrate        concentration in supernatant liquid 60; V_(x) is volume of fifty        percent sodium citrate to be added; and C_(0.50) is        concentration of fifty percent sodium citrate (i.e. 0.50).    -   Note that the desired end concentration of sodium citrate in        solution is 0.22 or twenty-two percent.

Solving for V_(x) yields Formula IV which may be used to calculatevolume of sodium citrate to be added:V _(x) =V ₆₀*(C _(e) −C ₆₀)/(C _(0.50) −C _(e))

-   -   As an example, for a volume of V₆₀ of 552 liters; a        concentration of C_(e) of 0.22; a concentration of 0.12 for C₆₀;        and a concentration of 0.50 for C_(0.50):        V_(x)=197 liters        After adding volume V_(x) of sodium citrate, stir for two to        four hours while retaining the temperature of this mixture        between two and eight degrees Centigrade. Note that this        solution will change color to a pale yellow as the additional        sodium citrate is added and the mixture is stirred.

After stirring, per step 66, centrifuge the mixture, use a continuousflow centrifuge while maintaining the temperature in the range of two toeight degrees Centigrade to separate paste 70 from supernatant 72. Theresultant supernatant (supernatant 72) contains essentially no IgG.Virtually all of the IgG of plasma 30 is now found in paste 70.

Reference is now made to FIG. 2 as point 80 continues to point 80′ forflow path portion 10-2 of flow path 10. Contents of paste 70 includesIgG, other serum proteins and sodium citrate. The sodium citrate must beremoved from paste 70 to permit IgG to be isolated by ion exchangechromatography. First, paste 70 is liquified using purified water (ofabout four times the volume of paste 70) as step 90. Product of step 90is an IgG rich solution 100. Initial conductivity of solution 100 isapproximately 20 milliSiemens/centimeter (mS/cm).

Removal of sodium citrate is accomplished by continuous diafiltrationusing purified water as a solvent in step 102 which separates solution100 into removed sodium citrate 110 and desalted IgG retentate 112.Completion of step 102 is indicated when the conductivity of retentate112 is reduced to 500-900 microSiemens/centimeter (uS/cm). Fordiafilitration in step 102, a Millipore (or comparable) diafiltrationsystem equipped with 30 KD cut-off membranes may be employed.

Viral inactivation of IgG rich retentate 112, associated with step 120,may be accomplished as a double viral inactivation step involving afirst solvent/detergent (S/D) method, followed by an augmented S/Dmethod. The first method employs raising the temperature of retentate112 to approximately 27° Centigrade (temperature may range from 24-30°Centigrade). A sufficient volume of Triton X-100 or Tween 80 is thenadded to make a one percent solution and sufficient Tri-N-ButylPhosphate to make a three tenths of one percent solution to make a firstS/D added mixture. The first method continues by incubating the firstS/D added mixture at 27° Centigrade for three hours during which timelipid enveloped viruses are inactivated. From this point, procedurescurrently available, inactivation and fractionation processes may beemployed. However, a currently preferred process is hereafter providedfor completeness.

For step 120, a S/D concentrate may be made as follows:

-   -   Add 30 milliliters of Tri-N-Butyl Phosphate to 800 milliliters        of purified water. Mix well. Add 100 milliliters of either        Triton X-100 or Tween 80 to the mixed solution. Again, mix well        to provide a final, mixed S/D solution. Add enough purified        water to bring the total volume of the final mixed solution to        1000 milliliters. One more time, mix well. So made, the final        solution is a 10× concentrate. Add 100 milliliters of this        concentrate to each 900 milliliters of retentate 112 to form the        first S/D added mixture.

After three hours of incubation, add, to the solution resulting from thefirst S/D method, sufficient formaldehyde to make a three tenths of onepercent solution and sufficient phenol to make a three tenths of onepercent solution to form an augmented mixture to begin the augmentedmethod phase of step 120. Incubate at approximately twenty seven degreesCentigrade for an additional three hours, after which time non-envelopedand enveloped viruses are inactivated.

For step 120, an “augmented” concentrate may be made as follows:

-   -   Add 13.4 milliliters of thirty-seven and one-half percent        formaldehyde solution to 900 milliliters of purified water. Mix        well. Add fifty grams of phenol (reagent grade) to this mixture.        Again, mix well. Add enough purified water to bring the total        volume of the “augmented” preparation to one thousand        milliliters. Once more, mix well. This preparations contains        50,000 parts per million each of formaldehyde and phenol (five        percent of each). Measure the volume of the first S/D added        mixture. Add 167 milliliters of augmented concentrate to each        833 milliliters of first S/D added mixture to form the augmented        mixture.

Step 120 is completed by cooling the processed augmented mixture to atemperature of two to eight degrees Centigrade. So cooled, the augmentedmixture becomes IgG virus inactivated (VI) solution 122.

Alternatively, viruses may be removed by other methods (e.g.,chromatography, nanofiltration, pasteurization), if desired.

Step 124 involves use of column chromatography to remove viralinactivation chemicals. Such may be accomplished by the followingsub-steps:

-   -   1. Set up a short, wide column with Toyopearl CM-650C resin. The        Toyopearl resin is a weak cationic exchange resin used to        capture IgG in solution 122 while permitting other proteins from        solution 122 to flow through the column. It is important that        the conductivity of solution 122 be in a range of 100 to 900        microSeimens/centimeter (uS/cm). (It is preferable that such        conductivity is in a range of 400 to 600        microSeimens/centimeter.) IgG from plasma 30 binds to the        exchange resin in the low ionic strength solution.    -   2. Introduce solution 122 into the exchange resin at a slow        rate. Collect effluent liquid from the column and measure the        effluent liquid at 280 nano-meters in a one centimeter silica        cuvette in a high quality spectrophotometer. (As an example, a        Beckman DU-7 with a deuterium light source may be used.) It        should be noted that optical density of the effluent will        increase as proteins are introduced into the resin column.        Phenol in the viral inactivation solution (if used) also can        increase measured optical density. After all of solution 122 has        passed through the resin column and sterilants are washed from        the column, begin collecting the effluent when measured optical        density increases from its original value. A rise in optical        density is indicative of protein in the effluent. After a        period, optical density drops down to a level which is        indicative of little or no protein in solution. At this point,        collecting may cease. At this point, it is preferable to        thoroughly wash the resin with deionized water. Bound material        is IgG, identified along path 10-2 as bound IgG 130. Collected        effluent from the column includes all of the protein from plasma        30 except for IgG. This effluent is effluent solution 132. It is        recommended that serum protein electrophoresis be performed on        effluent solution 132 to confirm that little or no IgG has been        released into solution 132.

Depending upon size of the resin column, prepare a volume of two percentsolution of sodium chloride. Application of the sodium chloride is usedto effect release of attached IgG from resin particles. As is well-knownin chemistry art, a two percent solution is made by mixing 20 grams ofsodium chloride into one liter of deionized water. Sufficient volume oftwo percent sodium chloride solution should be made to equal about tentimes the volume of the resin column.

For step 134, add the sodium chloride solution to the column, collectingeffluent from the column. Concurrently, measure optical density of theeffluent solution at 280 nanometers using a spectrophotometer with a onecentimeter silica cuvette. Resultant optical density (OD) will be foundto suddenly increase as IgG is uncoupled from the resin and deliveredinto the effluent. Collect all high OD measured solution. When the OD ofthe effluent drops to a lower (normal) range, cease collecting thesolution. Resulting solution is IgG solution 140. Note that a high OD isindicative of protein content in solution, and that solution 140 maycontain small amounts of IgM and IgA, which requires further removal. Inaddition solution 140 contains sodium chloride which must be removedbefore any pure IgG can be isolated.

Reference is now made to continuation point 150 in FIG. 2 whichcontinues to continuation point 150′ in FIG. 3 for flow path portion10-3 of flow path 10. Sodium chloride is preferably removed fromsolution 140 by continuous diafiltration employing a diafiltrationsystem. Such may consist of a Millipore (or comparable) diafilitrationsystem equipped with 30 KD cut-off membranes. As performed in step 152,the diafilitration solvent is purified water. As may be noted, initialconductivity of solution 140 is approximately fiftymilliSiemens/centimeter (mS/cm). At completion of diafiltration,conductivity is reduced to 500-900 microSiemens/centimeter (uS/cm).

The products of diafilitration are an IgG rich retentate 160 and removedsodium chloride 164. It is recommended that serum electrophoresis beperformed at this step in the process to quantitate protein content inretentate 160.

Step 166 is a final step for purifying IgG rich retentate 160. For step166, it is preferred to set up a short, wide resin column with ToyopearlQAE-550C resin. Such resin provides a strong anionic exchange forcapturing other proteins in IgG rich retentate 160, while permitting IgGin solution to flow through the column. It is important thatconductivity of retentate 160 be in a range of 100 to 900microSeimens/centimeter, and preferably, within a range of 400 to 600microSeimens/centimeter. In this manner, IgG in retentate 160 will passthrough the resin column in Step 166 without binding, while otherproteins, including IgM and IgA, will bind to resin in the column andthus be removed from solution. In this manner, any contaminatingresidual proteins 170 are effectively separated from a purified IgGsolution 172.

As the process is continuous, it is recommended that IgG solution 172 becollected and the OD measured for a target 280 nm. Collect the high ODeffluent solutions. When the measured OD drops, cease collecting. Thepooled solution is relatively dilute.

The pooled solution is concentrated using step 180 via ultrafiltration.For such ultrafiltration, a hollow fiber filter may be used, or aMillipore ultrafiltration system (Pellicon) or equivalent, (10K to 30Kdalton retentation) to concentrate to a twelve percent IgG solution 182.Excess water 184 is removed in the process of step 180. The resultingtwelve percent concentrate should have only a trace amount of sodiumchloride and the pH should be approximately seven. Conductivity shouldmeasure about 100 to 900 microSiemens/centimeter.

To stabilize the twelve percent IgG solution 182, add (step 190) amaltose or sorbitol solution to dilute the twelve percent solution toexactly ten percent. The final ten percent solution (IgG solution 192)should contain approximately five percent maltose or sorbitol (whicheveris used).

Optionally, to remove viruses 204 from IgG solution 192, nanofiltrationmay be performed by passing the ten percent solution 192 through a virusretaining membrane (step 200) to produce a nanofiltered concentrate 202.

Reference is now made to continuation point 210′ in FIG. 4, for flowpath portion 10-4 of flow path 10, which continues from continuationpoint 210 in FIG. 3. As is standard procedure, (depending upon executionof prior option step 200) either stabilized IgG concentrate 192 ornano-filtered IgG concentrate 202 is diluted with deionized water instep 220 to produce a bulk purified IgG solution 222. Contaminatingbacteria may be removed by passing solution 222 through a sterilizingfilter in step 230 to produce a sterilized bulk IgG solution 232.Removed contaminating bacteria 234 may be disposed of by methodscurrently known in the art.

Resulting sterile solution 232 may be filled into vials per standardprocedures in step 240 to produce a lot 242 of vials of solution 232. Asrequired for quality assurance, final testing and inspection of lot 242may be made in step 244 in cooperation with step 246 to produce a lot250 of validated vials of solution 232, with any discard 252 beingremoved therefrom.

Reference is now made to FIG. 1A wherein flow chart 10-1′ disclosesoptional steps, for separating out a paste from which Factor VIII, vonWillebrand Factor, and other clotting factors may be fractionated, areseen. Steps seen in FIG. 1A which differ from steps seen in FIG. 1 areoptional and may be followed to produce a cryoprecipitate from whichFactor VIII, von Willebrand Factor, and other clotting factors can beremoved. In the case of the process flow seen in FIG. 1A, procedure 40′is defined to gradually warm plasma 30 to zero to four degreesCentigrade. Such warming results in a thawed plasma in which acryoprecipitate is suspended. Per step 42, the thawed plasma iscentrifuged to yield the cryoprecipitate in the form of a paste 44.Paste 44 may be subsequently separated and processed by known methods toprovide Factor VIII, von Willebrand Factor, and other clotting factors.The remaining separated material, named cryo-poor plasma 50′, is thenprocessed in the same manner as prepared plasma 50, as disclosed supra.

Results of a Fractionation Procedure Performed According to the InstantInvention:

In order to show the efficacy and precision of separation of steps ofthe instant invention disclosed herein, the following results have beenextracted from a laboratory report, dated Aug. 8, 2005.

In the procedure, fresh frozen human plasma was used. As is typical insuch procedures, a pool was made from four to eight bags of thawedplasma (see step 40, FIG. 1). Commercial equipment available fromBeckman-Coulter was used to evaluate various fractions as they becameavailable. The Beckman, “Appraise”, densitometer was used to scan theBeckman agarose gels for serum protein electrophoresis as part of aParagon Electrophoresis System. For each fraction made from the pool,between three and five gel slits were loaded with five microliters ofproduct. Results were averaged to obtain a better representative resultfor each fraction. Such results are found in tables provided hereafter.

The gels were electrophoresed for twenty-five minutes at 100 VDC at a pHof 8.6 and later stained with a Paragon blue stain. The Appraisedensitometer was used to scan the stain-dried gels at a wavelength of600 nanometers twice for each gel slit (ten gel slits per gel wereused). An average graphic representation of the distribution of fivedifferent protein fractions, based upon density of attached dye as wellas a numeric presentation of each fraction was derived. The numericpresentation was based upon a computer analysis of peaks and valleys ofgenerated graphs at selected locations within the gel pattern asoccurred between the anode and cathode on each gel. Presentation valueswere totaled and dye percentage was divided by the total dye amount toprovide a percentage for evaluation. Note that the grand total, summingeach individual blood fraction always equals one hundred percent.

As seen in Table I below, the five different protein fractions areidentified as: Albumin, Alpha 1, Alpha 2, Beta and Gamma globulin.Before fractionation, a sample was removed from the pool andelectrophoresed to determine the average fractional values of eachfraction before beginning fractionation. Representative results for theaverage base pool material are listed below as percentages of wholeplasma: TABLE I Percentage content of each fraction Albumin Alpha 1Alpha 2 Beta Gamma Base plasma 61.2 7.1 9.7 13.0 8.9

Plasma (i.e., plasma 50) from the pool was treated with the addition ofa volume of fifty percent sodium citrate to a volume of plasma to make atwelve percent solution of sodium citrate (step 54). This mixture wasstirred for sixty minutes at two to eight degrees centigrade and wasthen centrifuged (Step 56) for sixty minutes at two to eight degreescentigrade. The resulting supernatant solution 60 was measured. Theremaining paste 62 was weighed and put into solution by addition ofdeionized water. The two solutions (60 and 62 (dissolved)) wereelectrophoresed using procedures cited supra, the results of which aresummarized in Table II, below: TABLE II Resulting percentageconcentrations Albumin Alpha 1 Alpha 2 Beta Gamma 12% Paste 62 67.8 2.79.1 20.4 nd* (dissolved) 12% Supernatant 28.6 1.5 10.7 40.3 16.1 60*nd = none detectedThere was no gamma globulin found in Paste 62 (dissolved). However,there was gamma globulin found in Supernatant 60.

Next, sufficient fifty percent solution sodium citrate was added tosupernatant 60 (step 64) to obtain a final mixture that containedtwenty-two percent sodium citrate. This solution was also stirred forsixty minutes at two to eight degrees centigrade. After centrifuging(see step 66) for sixty minutes at two to eight degrees centigrade, theresulting supernatant solution 72 was measured. The remaining paste 70was weighed and put into solution (step 90) by the addition of deionizedwater (four times weight of paste 70 in milliliters) to form IgG richsolution 100. Samples of supernatant 72 and IgG rich solution 100 wereelectrophoresed by the procedure cited supra, the results of which aresummarized in Table III, below: TABLE III Percentage concentrations ofindicated solutions Albumin Alpha 1 Alpha 2 Beta Gamma 22% Supernatant82.4 13.9 3.6 nd* nd* 72 22% Sol. 100 16.7 1.3 10.5 32.5 39.0(dissolved)*nd = none detectedThere was no gamma globulin found in supernatant 72. However, there wasgamma globulin found in IgG rich solution 100.The 22% supernatant fluid (which contained mostly albumin) containedessentially no beta and gamma globulin (i.e., none of such that wasdetected). The twenty-two percent paste solution contained the gammaglobulin of interest for further fractionating to produce intravenousgamma globulin for injection. Note also that, in step 90, time should beallowed for the paste to solvate prior to performing electrophoresis. Inthe experimental process, a plasma fraction between twelve percent andtwenty-two percent sodium citrate was selectively isolated out for usein this isolation procedure.

To remove sodium citrate (step 102) trapped in the twenty-two percentpaste solution, a Pellicon unit was selected to diafilter solution 100.On the average, about seven times the volume of solution 100 wasrequired to diafilter the sodium citrate and bring conductivity of theresulting solution down to a range between 400 and 800microSiemens/centimeter (uS/cm), before performing any column work.

After diafiltration, the desalted protein solution 112 was treatedelectrophoretically to determine any changes or losses as a result ofdiafiltration step 102. Because sodium citrate was removed, proteinmovement in the electrophoretic pattern was changed somewhat throughlack of interference with a contained salt. The resulting pattern wassomewhat longer than a high salt concentration pattern. This elongatedpattern allowed gamma globulin to separate more readily from betaglobulin with a resulting increase in measured percentage as seen inTable IV, provided below: TABLE IV Percentage content of diafiltratefractions Albumin Alpha 1 Alpha 2 Beta Gamma Solution 112 16.6 1.5 9.926.9 45.2As seen in Table IV, approximately forty-five percent of solution 112was gamma globulin and solution 112 exhibited better separation in theelectrophoresis pattern. Note, that the beta fraction went down withbetter separation in the electrophoresis pattern.

At this point, various currently employed methods could have been usedto purify the gamma globulin in solution 112. For that reason, thecompletion of this experiment could have varied from steps seen in FIGS.2-4. In the case of this experiment, the solution was first treated witha Solvent/Detergent solution of three hours at twenty-seven degreesCentigrade. Then an augmented sterilization solution, performedaccording to U.S. Pat. No. 6,881,573, titled AUGMENTED SOLVENT/DETERGENTMETHOD FOR INACTIVATING ENVELOPED AND NON-ENVELOPED VIRUSES, issued toAllan L. Louderback, filed Sep. 12, 2003, was added to the mixture andfurther incubated for an additional three hours at twenty-seven degreesCentigrade. This dual inactivation treatment of the dialyzed diafilteredsolution inactivates both enveloped and non-enveloped viruses.

The sterile treated solution was transferred to an ion exchange columnloaded with Toyopearl CM-650C resin. The resin adsorbed gamma globulinand allowed all of the other proteins present in solution to flow out ineffluent from the column. After adding the solution to the column andadjusting the column flow to slowly drip out through the effluent end,effluent solution was measured at 280 nanometers to determine when allfree proteins and sterilants had been transported through the column.Afterward, the column was washed with a two times volume of deionizedpurified water to assure that the effluent has a very low measuredoptical density at 280 nanometers.

A two percent solution of sodium chloride was then dispensed onto thetop of the column and allowed to percolate through the column. Gammaglobulin which was adsorbed by resin particles was freed to flow out ofthe column into a receiving vessel.

Collected effluent from the column with purified water (labeled asPurified Water) and effluent from the column with the two percentsolution (labeled as two percent NaCl) were tested electrophoreticallyto show the result of selected isolation and release of gamma globulinfrom the resin particles. Results of this step is summarized in Table V,seen below: TABLE V Resulting percentage fractions Albumin Alpha 1 Alpha2 Beta Gamma Purified Water 26.0 2.7 15.2 57.0 nd* Two Pecent NaCl nd*nd* nd* 1.9 98.1*nd = none detectedNote that more than 98% of the gamma globulin was isolated in the firstresin treatment. The value for beta globulin of 1.9% may be the resultof an application spot when applying solution to gel. The two percentsodium chloride solution contained the gamma globulin (IgG) and,perhaps, with larger pools of plasma, may contain some IgA and IgMglobulins which should be removed.

The two percent sodium chloride solution was therefore diafiltered toremove the sodium chloride for a next column treatment. Diafilitrationwas again performed by passing the solution though a Pellicon unitwhereby the salt was removed, yielding a final product which had aconductivity of 400 to 800 microSeimens/centimeter (uS/cm). Note that itlikely takes about six volumes of dionized purified water to diafilterthe two percent solution.

As a final step, a column was filled with Toyopearl 560-C resin and thedesalted solution was added to the top of the column and allowed toslowly percolate through the column. In this column, gamma globulinflowed right through the resin and all other proteins attached to theresin (e.g., IgA and IgM) to yield a final effluent from the column(solution 172) that was 100% gamma globulin in an aqueous base. Theeffluent tested is seen in Table VI below: below: TABLE VI Percentagecontent of final solution Albumin Alpha 1 Alpha 2 Beta Gamma Solution172 nd* nd* nd* nd* 100*nd = none detected

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiment is, therefore, to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are, therefore, intended to be embracedtherein.

1. A process for blood-based material fractionation for producing auseful, non-denatured intravenous immunoglobulin (IgG) productcomprising the steps of: (a) acquiring a predetermined quantity ofblood-based material for processing; (b) preparing said quantity ofblood-based material for processing; (c) preparing for a firstfractionation step by adding a first predetermined quantity of a firstpredetermined concentration of sodium citrate to said quantity to form afirst separable product and a first residual product, said firstseparable product being substantially free of euglobulins; (d)separating said first separable product from said first residualproduct; (e) performing a second fractionation step by adding a secondpredetermined quantity of a second predetermined concentration of sodiumcitrate to said separated first separable product to form a secondseparable product and a second residual product; (f) separating saidsecond separable product from said second residual product; (g)preparing said second separable product for diafiltration; and (h)diafiltering said second separable product to form a lower volume thirdproduct substantially free of sodium citrate.
 2. The process forblood-based material fractionation according to claim 1 wherein step (a)comprises selecting a quantity of plasma as the blood-based material. 3.The process for blood-based material fractionation according to claim 1wherein step (a) comprises selecting a quantity of a blood fractionationproduct as the blood-based material.
 4. The process for blood-basedmaterial fractionation according to claim 1 wherein step (b) comprisesthawing said quantity of blood-based material.
 5. The process forblood-based material fractionation according to claim 1 wherein step (c)comprises adding a fifty percent sodium citrate solution to saidquantity of blood-based material.
 6. The process for blood-basedmaterial fractionation according to claim 5 wherein step (c) comprisesforming said first separable product as a first supernatant solution,having a first predetermined sodium citrate concentration, incombination with said first residual product as a first paste residualproduct, having the same predetermined sodium citrate concentration. 7.The process for blood-based material fractionation according to claim 6wherein step (c) comprises forming said first separable product as afirst supernatant solution, having a twelve percent sodium citrateconcentration, in combination with said first residual product as afirst paste residual product, having the same twelve percent sodiumcitrate concentration.
 8. The process for blood-based materialfractionation according to claim 6 wherein step (c) comprises formingsaid first separable product as a first supernatant solution, having asodium citrate concentration in a first range between ten and fourteenpercent, in combination with said first residual product as a firstpaste residual product, having a sodium citrate concentration in thesame first range.
 9. The process for blood-based material fractionationaccording to claim 6 wherein step (c) comprises forming said firstseparable product as a first supernatant solution, having a sodiumcitrate concentration in a second range between eleven and thirteenpercent, in combination with said first residual product as a firstpaste residual product, having a sodium citrate concentration in thesame second range.
 10. The process for blood-based materialfractionation according to claim 1 comprising an additional step offractionating said first residual product into a group of productscomprising blood factors comprising Factors VIII, IX, and vonWillebrand, and fibrinogen.
 11. The process for blood-based materialfractionation according to claim 1 wherein step (d) comprises separatingsaid first separable product from said first residual product bycentrifuging.
 12. The process for blood-based material fractionationaccording to claim 11 wherein step (d) comprises removing said firstseparable product as a first supernatant fluid.
 13. The process forblood-based material fractionation according to claim 12 wherein step(e) comprises adding a fifty percent sodium citrate solution to saidfirst supernatant fluid.
 14. The process for blood-based materialfractionation according to claim 13 wherein step (e) comprises forming asecond paste product, having a second predetermined sodium citrateconcentration, and a second supernatant product having the same secondpredetermined sodium citrate concentration.
 15. The process forblood-based material fractionation according to claim 14 wherein step(e) comprises forming a second paste product, having a twenty-twopercent sodium citrate concentration, and a second supernatant producthaving the same twenty-two percent sodium citrate concentration.
 16. Theprocess for blood-based material fractionation according to claim 14wherein step (e) comprises forming a second paste product, having asodium citrate concentration within a range of twenty to twenty-fourpercent, and a second supernatant product having the same range oftwenty to twenty-four percent sodium citrate concentration.
 17. Theprocess for blood-based material fractionation according to claim 14wherein step (e) comprises forming a second paste product, having asodium citrate concentration within a range of twenty-one totwenty-three percent, and a second supernatant product having the samerange of twenty-one to twenty-three percent sodium citrateconcentration.
 18. The process for blood-based material fractionationaccording to claim 14 comprising an additional step of fractionatingsaid second residual product into a group of products comprising albuminand alpha-1-antitrypsin.
 19. The process for blood-based materialfractionation according to claim 14 wherein step (f) comprisesseparating said second separable product from said second residualproduct by centrifuging.
 20. The process for blood-based materialfractionation according to claim 14 wherein step (f) comprisesseparating said second separable product from said second residualproduct by filtering.
 21. The process for blood-based materialfractionation according to claim 14 wherein step (f) comprisesseparating said second separable product as a paste of a thirdpredetermined concentration having a resulting weight.
 22. The processfor blood-based material fractionation according to claim 21 whereinstep (g) comprises forming a liquid dilution of said separable pasteproduct of a third predetermined concentration by combining saidresulting volume of said separable paste product with a volume of waterhaving four times the resulting weight.
 23. The process for blood-basedmaterial fractionation according to claim 22 wherein step (h) comprisespassing the liquid dilution over a filtering membrane for the purpose ofseparating a portion of the third product from the added sodium citrateto provide the substantially free of sodium citrate third product. 24.The process for blood-based material fractionation according to claim 22wherein step (h) comprises passing the liquid dilution over a 30 KDdiafiltering membrane for the purpose of separating a portion of thirdproduct from the added sodium citrate to provide the substantially freeof sodium citrate third product.
 25. The process for blood-basedmaterial fractionation according to claim 23 wherein step (h) comprisestesting conductivity of the third product for a first predeterminedconductivity prior to diafiltration and testing the substantially freeof sodium citrate third product for a second predetermined conductivity.26. The process for blood-based material fractionation according toclaim 25 wherein step (h) comprises testing the conductivity to assurethird product conductivity is in the range of 500 microSiemens/cm(uS/cm) plus or minus 400 microSiemens/cm (uS/cm).
 27. The process forblood-based material fractionation according to claim 25 wherein step(h) comprises testing the conductivity to assure the substantially freeof sodium citrate third product conductivity is in the range of 500microSiemens/centimeter (uS/cm) plus or minus 400microSiemens/centimeter (uS/cm).
 28. The process for blood-basedmaterial fractionation according to claim 27 comprising the followingadditional step: (i) adding a predetermined volume of a predeterminedconcentration of detergent selected from a group of detergentscomprising a solvent detergent and an augmented solvent detergent forthe purpose of inactivating viruses contained in the substantially freeof sodium citrate third product to produce an IgG viral inactivatedsolution.
 29. The process for blood-based material fractionationaccording to claim 1 comprising a following additional step of coolingthe IgG inactivated solution to a range of two to eight degreesCentigrade.
 30. The process for blood-based material fractionationaccording to claim 29 comprising the following additional steps: (j)performing column chromatography on the IgG viral inactivated solutionto produce a resin bound IgG and an effluent; (k) separating the resinbound IgG from the effluent; and (l) washing the column resin bound IgGwith a predetermined concentration of sodium chloride to produce an IgGrich solution free of viral inactivation chemicals.
 31. The process forblood-based material fractionation according to claim 30 wherein step(j) comprises using a weak cationic exchange resin for the purpose ofpurifying the IgG rich solution free of viral inactivation chemicals toproduce a purified IgG solution.
 32. The process for blood-basedmaterial fractionation according to claim 30 wherein step (j) comprisesusing a Toyopearl CM-650C resin.
 33. The process for blood-basedmaterial fractionation according to claim 30 wherein step (j) comprisestesting conductivity of the IgG rich solution free of viral inactivationchemicals to assure said solution has a conductivity in the range of100-900 microSiemens/centimeter (uS/cm).
 34. The process for blood-basedmaterial fractionation according to claim 30 comprising a followingadditional step: (m) concentrating the purified IgG solution to apredetermined concentration of IgG solution by ultrafiltering saidsolution.
 35. The process for blood-based material fractionationaccording to claim 34 wherein step (m) comprises concentrating thepurified IgG solution to a twelve percent concentration of IgG solutionby ultrafiltering said solution.
 36. The process for blood-basedmaterial fractionation according to claim 34 comprising a followingadditional step: (n) stabilizing the predetermined concentration of IgGsolution by adding a stabilizing chemical selected from a group ofchemicals including maltose and sorbitol to produce a stabilized IgGconcentrated solution.
 37. The process for blood-based materialfractionation according to claim 36 comprising a following additionalstep: (o) if desired, nanofiltering the stabilized IgG concentratedsolution to produce a nanofiltered stabilized IgG concentrated solution.38. The process for blood-based material fractionation according toclaim 37 comprising a following additional step: (p) using sterile waterfor injection, diluting the stabilized IgG concentrated solution to apredetermined concentration of bulk purified IgG.
 39. The process forblood-based material fractionation according to claim 36 comprising afollowing additional step: (q) using sterile water for injection,diluting the stabilized IgG concentrated solution to a concentration often percent bulk purified IgG.
 40. The process for blood-based materialfractionation according to claim 38 comprising a following additionalstep: (r) filtering for the purpose of sterilizing the bulk purified IgGconcentrated solution.
 41. The process for blood-based materialfractionation according to claim 40 comprising a following additionalstep: (s) dispensing the sterilized bulk purified concentrated IgGsolution into sterile vials for distribution.
 42. The process forblood-based material fractionation according to claim 41 comprising afollowing additional step: (t) testing samples of contents of thesterile vials for safety and efficacy and, when vials pass predeterminedtesting criteria, releasing the vials to distribution channels.
 43. Theprocess for blood-based material fractionation according to claim 1wherein a further step following step (f) processes said second residualproduct to separate out albumin and alpha-1-antitrypsin.
 44. The processfor blood-based material fractionation according to claim 1 wherein saidpreparing of said quantity of blood-based material comprises a furtherstep of obtaining a cryoprecipitate by freezing blood plasma followed bycontrolled thawing at zero to four degrees centigrade to form aprecipitate in a liquid suspension and separating out the precipitate toprovide a resultant quantity of blood-based material.
 45. A method forseparating euglobulins from blood elements being fractionated fromblood-based material for the purpose of performing a subsequentdiafiltration procedure on the separated blood elements comprising thesteps of: (a) acquiring a predetermined volume of blood-based materialfor processing; (b) preparing said volume of blood-based for processing;(c) preparing for a first fractionation step by adding a firstpredetermined volume of a first predetermined concentration of sodiumcitrate to said sample to form a first separable supernatant product anda first residual product, said first separable supernatant product beingsubstantially free of euglobulins.